Cytokinin Signaling
The plant hormone cytokinin is involved in many developmental processes and plays a critical role in numerous physiological responses to changes in the environment (Mok and Mok, 2001). In recent years significant progress has been made towards the understanding of how the cytokinin signal is perceived and transduced (Ferreira and Kieber, 2005; Grefen and Harter, 2004; Heyl et al., 2006; Hwang et al., 2002; Hwang and Sakakibara, 2006; Kakimoto, 2003; Mizuno, 2004). In the current model, which has been mainly developed in Arabidopsis, the hormone is perceived by membrane-bound hybrid histidine kinase receptors (AHKs), which auto-phosphorylate upon binding of the hormone ligand. After trans-phosphorylation within the receptor, the phosphoryl residue is transferred to a histidine phospho-transfer protein (AHPs), which subsequently locates to the nucleus, where it activates B-type response regulators (ARRs) via phosphorylation. These transcription factors activate the transcription of their target genes, one group of which are the A-type response regulators. A negative feedback on the cytokinin signaling pathway was shown to be mediated by members of this protein class (Hwang and Sheen, 2001; To et al., 2004).
B-type response regulators are characterized by the presence of Myb-class DNA binding domain, called GARP domain, in addition to the response regulator domain. Several experiments have shown that B-type ARRs can bind to DNA and activate the transcription of their target genes in response to cytokinin treatment (Hosoda et al., 2002; Hwang and Sheen, 2001; Imamura et al., 2003; Lohrmann et al., 2001; Sakai et al., 2000). RT-PCR and promoter-GUS fusion experiments have demonstrated that the members of the B-type ARR family have large and overlapping expression domains (Mason et al., 2005; Tajima et al., 2004). The analysis of B-type ARR mutants has revealed their involvement in cytokinin signaling, but also a high level of functional redundancy (Hass et al., 2004; Horák et al., 2003; Mason et al., 2005; Sakai et al., 2001). Mason et al. (2005) studied single, double and triple mutants of ARR1, ARR2, ARR10 and ARR12 in various combinations (Mason et al., 2005). Different cytokinin response assays showed an increasing cytokinin resistance for higher order mutants. Surprisingly, beside a longer primary root in some mutants no strong morphological alterations were detected, which would be expected in case of a strong reduction of the cytokinin responsiveness. This could indicate that the degree of redundancy among B-type ARRs is even higher and/or that other transcription factors compensate for the loss of B-type ARRs. One other family of transcription factors which has recently been shown to be involved in mediating a cytokinin response are the cytokinin response factors (CRF)(Rashotte et al., 2006).
Chimeric Repressor Silencing Technology
The chimeric repressor silencing technology (CRES-T) has been developed to study the consequences of silencing the target genes of transcription factors and has also been used to overcome the experimental limitations caused by functional redundancy of transcription factor families. In 2001 Ohta and colleagues mapped a repression motif of transcriptional repressors of the class II ethylene response factors (ERF) which is both necessary and sufficient for the repression activity of the protein (Ohta et al., 2001). This so-called ERF-associated Amphiphilic Repression (EAR) motif or variations of it are found in numerous plant transcriptional repressors (Kazan, 2006; Ohta et al., 2001; Tiwari et al., 2004). The length and the repression potential of the EAR motif were improved resulting in the so-called SRDX motif (Hiratsu et al., 2003). Fusion of this motif to transcriptional activators converts them into dominant repressors (Hiratsu et al., 2003). Interestingly, these dominant repressors may repress not only the transcription of their own target genes, but also the expression of target genes of other members of their respective gene family. For example, the CUC1 and CUC2 transcription factors are functionally redundant and a loss of function phenotype is seen only in the cuc1 cuc2 double mutant plants, while mutation of both single genes does not cause a phenotype (Aida et al., 1997; Takada et al., 2001). The transgenic expression of a chimeric CUC1-SRDX gene induced the compound phenotype of a cuc1,cuc2 double mutant, demonstrating that its dominant-negative function encompassed CUC1 and CUC2 target genes (Hiratsu et al., 2003). The technology has not yet been used to investigate transcription factor gene families with numerous members.